In the last 50 years, two fields of study have converged on the protein calcineurin (CaN) because of it's pivotal role in signal transduction, particularly T-cell activation and memory development. CaN is ubiquitously produced with pleiotropic roles. It has been found that the physiological consequence of inhibition of CaN is sustained neurotransmitter release and eventual blockage of many cellular functions, including signal transduction pathways. See, Barford D. (1996) TIBS 21:407. With the improvement in organ transplant and the proliferation of the HIV virus, much attention has been focused on the immune system. While this system is an interesting physiological field by any counts, pharmaceutical companies also see the advantages in being able to control many immune response mechanisms. For example, inflammation is an immune response, one that can interfere with healing.
The other field is neurochemistry, where the study of memory has been found to be linked to differing potentiation rates of neurons during development. CaN appears to play an important role in calcium signaling and neural transmitter amplification as well as neuron development in infants. See, e.g., Quinlan et al. (1996) J Neuroscience 16:7627.
Since protein phosphorylation controls so many cellular events, regulation requires many levels. It must be controlled quite tightly in order to keep competing processes in balance. CaN offers a wonderful opportunity to observe many different regulatory mechanisms at work. These include site localization, Ca2+-activation, auto-inhibition (by auto-inhibitory domain—AID), CnB-activation, CaM-activation. In addition, there are a number of recent studies elucidating the great complexity of the regulation to which CaN is subject.
In understanding the regulation mechanism for Cn, the FKBP12-FK506-binding site is very important. It appears to be the same binding site for binding of cyclophilin-cyclosporin A(CyP-CsA) (found by genetic methods) and the site for binding of AKAP79. See, for example, Liu et al. (1992) Biochemistry 31:3896-3901; and Faux et al. JBC 272:17038. All three exhibit classical noncompetitive inhibition, suggesting a common mechanism. The suggested CaN-binding domain on AKAP79 is has high sequence similarity to residues 32-47 of FKBP12. It has also been found that a 22-residue peptide encompassing this FKBP12 sequence inhibits CaN by other than steric hindrance. Kawamura et al. (1995) JBC 270:15463.
Mutational studies of the b12/b 13 loop of yeast CaN results in CyP resistance, suggesting that these loops are important in binding the immunosuppressant complexes, and, perhaps, AKAP79. Wei and Lee recently showed that mutagenesis of the L7 loop (310-314+) connecting b-strands 12 and 13 has significant effects on activity. Wei et al. (1997) Biochemistry 36:7418. Both modification of the L7 loop and truncation of the C-terminus lead to the hyperactivitation of CaN. They also determined that the effects of mutation of the L7 loop are separable from the effects of deletion of the AID. Since mutations to L7 increase the catalytic efficiency of the enzyme towards pNPP, it must have something to do with changes in the active site. It is thought that CnA exists in a conformationally restrained state that is not completely relaxed by CnB and CaM. It has double inhibition to ensure that it only acts when needed. The mechanism of inhibition by AID is more than mere steric hindrance to binding.
One regulatory mechanism is that kinases and phosphatases are maintained at discrete cellular locations through their interaction with anchoring proteins. Enzymes may be positioned in close proximity to specific substrates, which then can be efficiently modified in response to the appropriate signals. Ser-Thr kinases and phosphatases are often maintained by scaffold proteins. In it's function in neuronal signaling, AKAP79 has been investigated in connection with CaN. Structural studies of CaN, AKAP79, and FKBP12 suggest that this regulation by inhibition is not merely by steric hindrance. Politino & King suggested in 1990 that it might function in membrane anchoring, from studies with phospholipid interactions (Politino et al. (1990) JBC 265:7619). Griffith has noted that other EF-hand superfamily proteins use Ca2+ as a switch, extruding the myristic acid upon Ca2+ binding to allow adherence to the cell membrane (Griffith et al. (1995) Cell 82:507).
There is an autoinhibitory domain (AID) at the carboxyl terminal of the CnA subunit. It lies over the substrate-binding channel of the catalytic domain. When CaN is auto-inhibiting, the CaM-binding domain, which is an amphipthic a-helix at the carboxyl terminus of the CnA, lies under the CnB-binding helix, linked at one end to the AID. This places the AID close to the active site where it could inhibit the binding of substrates and inhibitors. A Glu sidechain H-bonds with two of the metal-bound water molecules, sterically hindering substrate binding. See, for example, Stoddard et al. (1996) Curr Opin Struct Biol. 6:770. In human CaN this segment is Ser 469-Arg 486, with Glu 481-Arg-Met-Pro 484 making the most contact with the substrate-binding cleft. This segment consists of 2 short a-helical regions plus a 5-residue extension. It is missing from the bovine CaN-FKBP12-FK506 complex shown here.
Upon the addition of Ca2+, both CaM and CnB are activated. (Even without CaM, CnB confers some activation on CnA.) This activation apparently disrupts the interaction between CaM and the CnB-binding helix on CnA, moving the AID away from its inhibitory position(Barford D. (1996) TIBS 21:407)
The CnB-binding site on CnA is a long 22-residue a-helix. In 1995, Watanabe, et al. identified the CnB-binding helix as residues 328-390 (Watanabe et al. (1995) JBC 270:456). How CnB binds to CnA is very different from how CaM binds, even though they are very similar. The two domains of CnB can be superimposed by translation of 22 Å along the helix. The Kissinger structure of the human CaN structure (which is not yet available for download) has an additional CnA amino-terminal sequence that assists in CnB-binding. This sequence forms a part of the binding cleft for the carboxyl-terminal lobe of CnB. While there seems to be some conformational change in CnA upon the binding of CnB, it is not clear how the information is transmitted to the active site on the CnA subunit.
Klee et al. showed in 1988 that Ca2+/CaM activates CaN by increasing Vmax, whereas Ca2+ binding to CnB decreases Km and increases Vmax. See, for example, Klee et al. (1988) Adv. Enzymol. 61:149. Watanabe et al. in 1995 showed that the CnB-binding hydrophobic fragment is between the CaM-binding area and the active domain. This enabled Griffith to identify the subunit not present in the crystal structure to be the CaM-binding domain. Watanabe used a GST fusion protein expressed in Sf9 insect cells. A very highly conserved sequence on the CnA subunit was tested with site-directed mutagenesis, concluding that residues 349/350 and 356/357 Glu in the CnA are essential to binding CnB.
CaM and CnB apparently activate CnA by different but complimentary mechanisms. In the Kissinger structure, the predicted CaM-binding domain is quite disordered. It is thought to lie between residues 390-414. In contrast with CnB, CaM binds with the two domains on opposite sides of the helix related by a 2-fold rotation axis. Without that part of the crystal structure it would be interesting to model the interaction discussed in the literature. The primary sequence is available at Swiss-prot and adding the CaM-binding helix with the CaM docking might also help understand the mechanism of AKAP79-binding and cell localization better. For now, it is clear that how the myristoyl group is extruded by the coordinating structures is difficult to see since where the MYR group is linked to the CnB molecule is not definite.
CaN is not a huge protein, but it does offer great opportunities to observe the many ways such a small protein can exert broad influence. CaN is a heterodimer, with a 59-kDa CnA (catalytic) subunit and a 19-kDa CnB (regulatory) subunit. See, Cohen et al. (1989) JBC 264:21435.
At least 2 genes encoding isoforms of CnA have been identified from complimentary DNA cloning of the major catalytic subunit of CaN in mammalian brain. The a and b genes are localized on human chromosomes 4 and 10 respectively. A major difference between two isoforms was a long polyPRO helix (11 Pro) in b which may play a role in regulation (Zhuo et al. 1994) JBC 269:26234). The catalytic subunits of the other Ser-Thr phosphatases share the same gene family as CnA, sharing ˜40% sequence identity. There is an additional isoform in mammalian CnA with 54% identity (CaNw) that is similar around the CaM-binding domain but may have a distinct substrate specificity.
Only 1 gene for CnB, located on human chromosome 2 has been found (Navia (1996) Curr. Op. Struct. Bio. 6:838). Kawamura et al. (1995) JBC 270:15463 shows the primary sequence structure of the binding site of the subunits of the heterodimer on CnA. It also shows the parts of the molecule not found on the Griffith structure (dCnA).
Various domains have been identified on the CaN subunits. A distorted b-sandwich motif forms the core of the globular part of the enzyme. It includes most of the active-site residues, the metal-coordinating residues, and an auto-inhibitory domain. This globular domain is approximately 35 Å×35 Å×45 Å. A motif on the edge of this b-sheet sandwich coordinates the metal ions necessary for activity. See 5Stoddard et al., (1996) supra.
The mechanism of calcineurin and the other Ser/Thr protein phosphatases depends on the divalent metal coordinating site. The core structure of the globular domain is a central distorted b-sandwich of 11 b-strands surrounded on one side by seven a-helices and on the other by 3 a-helices and a three-stranded b-sheet. A shallow catalytic channel is created by the interface of the two b-sheets. Three parallel b-strands of sheet 1 constitute a mononucleotide-binding domain with the secondary structure organization b-a-b-a-b. The three invariant sequence motifs form loops connecting the carboxyl terminus of the b-strand with a-helices (6 Goldberg et al. (1995) Nature 376:745). These loops, together with those emanating from the carboxyl terminus of two b-strands of the opposite b-sheet, provide the catalytic residues. Zn2+ and Fe3+ are coordinated in this active site.
Zn2+ is coordinated by 1 Asn and 2 His side-chains. 2 Asp, 1 His, and 1 water coordinate Fe3+. Both metals have a coordinating oxygen from a bound phosphate in the CaN Griffith structure. The metal ions are located 3 Å apart in the active site, and have an Asp side chain that acts as a monodentate-bridging ligand between the metals. The bound phosphate in the structure could represent the labile phosphate in the dephosphorylation reaction. It is stabilized by interactions with guanidinium groups of two Arg residues and with the Ne2 of a single His residue. See, Griffith et al. (1995) Cell 82:507.
The CnB-binding site is a long 22-residue a-helix (sometimes called the b-binding helix: BBH). It is linked to the globular portion of the molecule by a short linker sequence. The CaM-binding site and the auto-inhibitory sites are missing in this structure, cleaved before crystallography. From structures of CaM and protein kinase II, an amphipathic a-helix is posited for the CaM-binding area of the molecule. It is after the CnB-binding helix.
CnB has 2 EF-hand Ca2+-coordinating domains. Each domain coordinates 2 Ca2+ atoms. The EF-hand motif consists of 2 a-helices joined by a b-loop. In this way, CnB is very similar to CaM, except without the long linking a-helix (8 Griffith, et al. (1995), supra). At the carboxyl end of the CnB molecule is a 14-carbon myristoyl residue, which recent studies link with regulation and cell-localization. See, Politino et al. (1990) JBC 265:7619.
The mechanism of calcineurin and the other protein phosphatases depends on the divalent metal coordinating site. The metal ions activate water molecules to catalyze hydrolysis of the phosphate in a single-step reaction. The mechanism is as follows: a metal-bound water attacks the phosphorus at in an SN2 nucleophilic mechanism. The metals act as Lewis acids to make water more nucleophilic and phosphorous more electrophilic. Histidine donates a proton to the oxygen leaving group from the Ser or Thr side chain (Barford (1996) TIBS 21: 407)
This is supported by studies showing that CaN cannot catalyze transphosphorylation reactions. See, Guerini (1997) Biochem. Biophys. Res. Comm. 235:271. Extensive studies show that no intermediates have been identified and the reaction must occur in a concerted manner (Barford D. (1996), supra). In studies of other similar PPases, purple acid phosphatase-mediated catalysis occurs with inversion of configuration, supporting SN2. In addition, Martin & Graves showed a pH dependence to CaN-mediated catalysis of pNPP; at pH 9 Vmax dropped precipitously, indicating that a monoanion is preferred as a substrate (Martin et al. (1994) Biochim. Et Bioph. Acta. 1206:136)
The activation of CaN by Mn2+ may be due to its substitution for Zn2+ or Fe3+. CaN is extremely similar to PP-1, which coordinates Mn2+ and Fe3+; Mn and Fe have similar atomic numbers See, Egloff et al. (1995) 254:942. However, according to Balinderan et al, ((1995) Molecular and Cellular Biochemistry 149/150:127 metal ions added to the solvent are probably responsible for a complex set of metal assisted equilibria and conformational transitions in CnA. In any case, this activation is most likely an artifact from in vitro studies.
As noted in discussing regulation, Wei and Lee recently showed that mutagenesis of the L7 loop (310-314+) connecting b-strands 12 and 13 has significant effects on activity. This clearly affects the active site, but how is not known. See, for example, Wei et al. (1997) Biochemistry 36, 7418-7424.